Genetic Screen in Drosophila Melanogaster Uncovers a Novel Set of Genes Required for Embryonic Epithelial Repair

Genetic Screen in Drosophila melanogaster Uncovers a Novel Set of Genes Required for Embryonic Epithelial Repair

Isabel Campos,1 Jennifer A. Geiger,1 Ana Catarina Santos,2 Vanessa Carlos3 and Antonio Jacinto4 Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal and Instituto Gulbenkian de Cieˆncia, 2780-156 Oeiras, Portugal Manuscript received September 25, 2009 Accepted for publication October 24, 2009

ABSTRACT The wound healing response is an essential mechanism to maintain the integrity of epithelia and protect all organisms from the surrounding milieu. In the ‘‘purse-string’’ mechanism of wound closure, an injured epithelial sheet cinches its hole closed via an intercellular contractile actomyosin cable. This process is conserved across species and utilized by both embryonic as well as adult tissues, but remains poorly understood at the cellular level. In an effort to identify new players involved in purse-string wound closure we developed a wounding strategy suitable for screening large numbers of Drosophila embryos. Using this methodology, we observe wound healing defects in Jun-related antigen (encoding DJUN) and scab (encoding Drosophila aPS3 integrin) mutants and performed a forward genetics screen on the basis of insertional mutagenesis by transposons that led to the identiﬁcation of 30 lethal insertional mutants with defects in embryonic epithelia repair. One of the mutants identiﬁed is an insertion in the karst locus, which encodes Drosophila bHeavy-spectrin. We show bHeavy-spectrin (bH) localization to the wound edges where it presumably exerts an essential function to bring the wound to normal closure.

OUND healing is essential to organisms through- of deep cells when those are exposed (see Jacinto et al. W out the animal kingdom. It must occur for 2001; Martin and Parkhurst 2004; Garcia-Fernandez restoring tissue integrity after injury both during et al. 2009 for review). embryonic and adult life. Epithelia, in particular, act Advances in live imaging of Drosophila embryos as a physical barrier protecting living organisms and expressing fluorescent proteins made time-lapse mi- their organs from the surrounding environment and croscopy of the epithelial healing process possible and have evolved robust mechanisms to ensure their the exact sequence of cell movements to be determined integrity. Simple embryonic epithelial tissues have an (Wood et al. 2002). The cells at the wound margin extraordinary capacity to reseal small discontinuities constrict their apical edges through the action of an very rapidly and efficiently through an epithelial re- actomyosin cable that assembles just minutes after sealing mechanism. This was initially described in the wounding and is linked intercellularly through adhe- chick embryo (Martin and Lewis 1992), but seems to rens junctions. Concomitant with the formation of the be conserved across species as it was shown to also occur purse string, cells at the wound margin begin to extend in mouse, frog, and fly embryos (McCluskey et al. actin-rich protrusions. When opposing wound margins 1993; Davidson et al. 2002; Wood et al. 2002). In all come into close proximity, filopodia and lamellipodia these systems, small epithelial wounds close via the from opposing flanks make contact and they appear to cooperation of three distinct mechanisms: the assembly pull the wound margins toward one another. For laser- of an actomyosin purse string in the epithelial cells at induced oval wounds of 10 by 20 mm, the entire the wound margin, the protrusive activity of epithelial healing process can be completed in just over 2 hours cells at the margin, and the contraction and ingression (Wood et al. 2002). The signaling cascades that regulate the epithelial resealing process are just beginning to be unraveled, but Supporting information is available online at http://www.genetics.org/ the known molecular mechanisms appear to be con- cgi/content/full/genetics.109.110288/DC1. served in both vertebrates and invertebrates, namely the 1These authors contributed equally to this work. involvement of Grainy-head (GRH) transcription factors 2Present address: Novartis Farma, 2710-444 Sintra, Portugal. or the JNK signaling cascade, transduced by JUN/FOS 3Present address: CRTD/DFG–Center for Regenerative Therapies Dres- transcriptional complexes (Ramet et al. 2002; Li et al. den, 01307 Dresden, Germany. 2003; Ting et al. 2003, 2005a,b; Galko and Krasnow 4Corresponding author: Instituto de Medicina Molecular, Faculdade de ace Medicina da Universidade de Lisboa, Edificio Egas Moniz, Av. Prof. Egas 2004; M et al. 2005). In the fly, the expression of some Moniz, 1649-028 Lisboa, Portugal. E-mail: [email protected] genes at the wound site is dependent on functional GRH

Genetics 184: 129–140 ( January 2010) 130 I. Campos et al. and JUN/FOS dimers (AP1) binding sites in their pro- ton Stock Center. The lines with a wound closure phenotype moter region (Mace et al. 2005; Pearson et al. 2009). were remapped by inverse PCR (iPCR) and sequenced, These observations are consistent with abnormal wound following protocols described at the Berkeley Drosophila Genome Center Web site (http://www.fruitfly.org/about/ healing in grh or basket/DJNK mutants’ larval cuticle and methods/inverse.pcr.html) and at the Bellen lab Web site the activation of JNK signaling pathway at wild-type larval (http://flypush.imgen.bcm.tmc.edu/pscreen/). We confirmed wound sites (Galko and Krasnow 2004; Mace et al. the insertion location as listed in FlyBase for all except 3 of our 2005). Recently, it was suggested that extension of actin- positive lines. Specifically, we could not determine the in- EP2475 c01955 f00677 based cellular processes by the wound-edge epidermal sertion locations for lines Eaf , Neu3 , and side .We did not succeed in remapping the Eaf EP2475 line by iPCR, but cells of Drosophila larvae is dependent on Pvr, a PDGF/ we assume the genomic location is correct since this line VEGF-like receptor, and one of its ligands, Pvf1 (Wu et al. failed to complement two different deficiencies in the region 2009). In addition, the Rho family of small GTPases (Df(2R)ED1673 and Df(2R)Drlrv28, stock nos. 9062 and 8888, including Rho, Rac, and Cdc42 are known to be critical to respectively). The Neu3c01955 line, which is listed as an insertion mediate the rapid cytoskeleton rearrangements that in the gene Neu3 (cytological map location 88C10–88D1, in 3R), was mapped by 59 primer sets (the 39 set did not work) to a control cell shape changes (as described above) of different location in the end of the 3L chromosome, in gene wound bordering epithelial cells during closure (re- CG40470. There are no available deficiencies in this region viewed in Jacinto et al. 2001; Martin and Parkhurst (the last three deficiencies of the chromosome all comple- 2004). The upstream signal activating the cells surround- mented the line, stock nos. 2587–2589) and this line comple- MB01428 ing the wound is still unknown, but it is established that ments another Neu3 allele (Neu3 , stock no. 23312) and a deficiency in the Neu3 region (Df(3R)ED10555, stock no. extracellular signal-regulated kinase (ERK) is phosphor- 23714). Therefore, we consider that the line Neu3c01955 is not an ylated upon wounding, an event required at wound sites insertion in the Neu3 region. Finally, the line side f00677, which is for a robust response (Mace et al. 2005). Taking together listed as an insertion in the gene sidestep (cytological map the fact that Drosophila GRH and FOS proteins can be location 97F6–97F10) was mapped by both 59 and 39 primer phosphorylated by ERK in vitro (Uv et al. 1997; Ciapponi sets to a different location, 86D9. This line fails to complement Df(3R)ED5516 (deletion of 86D8–86E13), confirming our et al. 2001) and more recent data identifying Stitcher, a mapping results. receptor tyrosine kinase that also induces ERK phos- For the pilot screen, the following mutant alleles were used: phorylation as a Grh target, one can envision a Grh- Jra1 (Kockel et al. 1997), puc E69 (Ring and Martinez Arias dependent positive feedback loop that could function as 1993), shark1 (Fernandez et al. 2000), tkv8 (Nellen et al. 1994), 1 rora 1 oung 2 rank an amplification mechanism ensuring efficient epider- shn (A et al. 1995), zip (Y et al. 1993), ush (F and Rushlow 1996), Pkn06736 (Spradling et al. 1999), scb5J38 mal wound repair (Wang et al. 2009). (Schock and Perrimon 2003), Rho11B (Magie and Parkhurst To gain new insights into the cell biology of epithelial 2005), fas2 (Lekven et al. 1998), and Egfr t1 (later renamed Egfr1) resealing, we performed a genetic screen using the (Price et al. 1997). Drosophila embryo with the aim of finding new genes All lethal or semilethal lines were crossed to balancer stocks involved in the regulation of wound healing. For that that drive eGFP under the twist promoter, active at embryonic purpose, we developed a wounding assay that facilitates stages. Specifically, males from original lines were crossed to either Gla/CyO-CTG or Dr/TM3-TTG virgins, depending on large-scale screening and validated it by showing that insertion site. Flies of the following generation were selected Jra, a mutant in the JNK signaling pathway, and scab,a against Gla or Dr and used to start new GFP-balanced stocks, mutant in an a-integrin isoform, are both required for which were then used to collect the screened embryos. embryonic wound healing. We then tested 655 piggy- OrR, ubi-DE-Cad-GFP (Oda and Tsukita 2001), and sGMCA, Bac and P-element insertion mutations (Exelixis) and which expresses the actin binding domain of moesin fused to GFP, labeling filamentous actin (Kiehart et al. 2000) were were able to identify 30 lines with impaired wound used as control lines. healing. One isolated mutant is an insertion in the karst Using standard strategies, the following recombinant stocks f05607 gene, encoding the Drosophila homolog of bHeavy-spectrin. were generated: CG2813 , ubi-DE-cad-GFP/CyO-CTG; Karst has been previously implicated in cytoskeleton CG5198c07150, ubi-DE-cad-GFP/CyO-CTG; CG5640 f01321,ubi-DE-cad- d11183 organization and associated with tissue morphogenesis GFP/CyO-CTG; and karst , sGMCA/TM6b-GFP. homas arnescu homas Wounding assay: Two-hour egg collections of GFP-balanced (T et al. 1998; Z and T 1999) but lines (Mutation/CyO-CTG or Mutation/TM3-TTG) taken on its precise function has remained elusive. We further standard apple juice agar plates supplemented with yeast show that the Karst protein accumulates around the extract, were allowed to develop at 18° overnight. Embryos wound edges in a cable-like manner, where it must play were dechorionated in bleach and typically 40–80 non-GFP an important function in the healing process. (homozygous mutant) stage 15/16 embryos were sorted under UV light. Homozygous mutant embryos were aligned ventral side up, stuck to double-sided tape on a slide, covered with halocarbon oil 700 (Sigma) and a coverslip, and subjected to MATERIALS AND METHODS wounding using a nitrogen laser-pumped dye laser connected to a Zeiss Axiovert 200M microscope (Micropoint Photonic Fly strains and genetics: A total of 655 insertional mutant Instruments). After wounding, the coverslip was removed and lines on the second and third chromosomes were picked from the embryos were left to develop at 22° for 16 hr before being the 2100 inserts chosen as single gene tags for the Genome scored under a dissecting scope for wound closure. The Disruption Project, originally generated by Exelixis Corpora- wound healing phenotype was calculated as a percentage of tion (Thibault et al. 2004) and distributed by the Blooming- nearly hatching first instar larvae with unclosed wounds over Drosophila Wound Healing Screen 131 the total number of wounded larvae (dead animals were disregarded for this calculation). Imaging: Live embryos were wounded as described above and live imaging was performed using LSM510 Meta confocal system (Carl Zeiss MicroImaging). Images were taken every 3 or 30 min. All images were processed using ImageJ imaging software (National Institutes of Health) and Photoshop (Adobe). Live wounded larvae were imaged using a DC500 Leica camera mounted on an upright widefield DM5000B Leica microscope under phase contrast conditions. Immunohistochemistry of wounded embryos: Stage 14 OrR embryos were selected and subjected to the wounding assay, except wounds induced were smaller and embryos were allowed to heal in a humid chamber for 1 hr before further processing. Wounded embryos were loosened from the tape with forceps and then removed from the oil with a paintbrush dipped in heptane (Sigma) and transferred to a glass vial containing fix mix, 1:1 heptane: 3% FA in PLP (3% formalde- hyde, 0.01 NaIO4, 0.1 m PIPES, pH 7.3, 0.1 m lysine) (modified from Thomas and Kiehart 1994), and incubated on a roller for 40 min at room temperature (RT). Embryos were removed and hand devitilinized in PBS (NaCl 137 mm, KCl 27 mm, KH2P04 43 mm, NaHP04-2H2047mm), incubated 1 hr in block (0.3% BSA in PBST, which is 0.3% Triton X-100 in PBS), and then overnight at 4° with the following primary antibodies diluted in block: m-armadillo at 1:50 (DSHB) and bH-specific antiserum no. 243 at 1:500 (Thomas and Kiehart 1994). Embryos were then rinsed three times and washed 1 hr in block, incubated 1 hr in secondary antibodies (Molecular Probes), diluted 1:200 in PBST (Alexa 568 anti-rabbit and Alexa 488 anti-mouse or Alexa 488 anti-rabbit and 1 mg/ml Alexa 594-phalloidin), rinsed three times and washed 1 hr in PBST, rinsed in PBS, and mounted in 80% glycerol with 2% Dabco.

RESULTS Wounding strategy and assay validation: To identify novel Drosophila strains with wound healing phenotypes, we designed and optimized a wounding assay suitable for a high-throughput screen (see materials and methods and Figure 1). To verify that the wounding assay is sensitive enough to ﬁnd genes required for wound healing, we tested candidate genes previously shown to be required either for wound healing or for dorsal closure (DC) in Drosophila (Figure 2). DC is a developmental process involving epithelial sheet movements to close a naturally occurring dorsal hole created when the germ band retracts after its extension. It has igure previously been reported that this process occurs in a F 1.—Wound healing protocol. (A) Two-hour egg collections from GFP-balanced lines are left to develop until mechanistically analogous way to embryonic wound stage 15/16 and homozygous, non-GFP, mutant embryos healing ( Jacinto et al. 2000; Wood et al. 2002). are selected under ﬂuorescent light for wounding. (B) Se- Therefore, we tested members of major pathways that lected embryos are mounted for laser wounding on a micro- control DC such as Jun-related antigen ( Jra), puckered scopic slide with the ventral side up. (C) Embryos are laser (puc), and Src homology 2 ankyrin repeat tyrosine kinase wounded on the medial ventral region. (D) After approximately 16 hr in a humid chamber, nearly hatching larvae (shark) in the DJNK signaling pathway and thickveins are screened under the dissection scope for wound closure (tkv), schnurri (shn), and zipper (zip) in the TGF-b and percentage of unclosed wounds calculated; arrows point signaling pathway. Furthermore, we tested additional to typical healed (left) and unhealed (right) wounds (see ma- genes involved in DC such as u-shaped (ush), Epidermal terials and methods for details). growth factor receptor (Egfr), Protein kinase related to protein kinase N (Pkn), scab (scb), and Rho1, which has also been 132 I. Campos et al.

Figure 2.—Dorsal closure mutants with wound healing phenotypes. (A) List of tested alleles, respective molecular function, and cellular process as in FlyBase (Tweedie et al. 2009). Wound healing phenotypes are shown as percentage of total (n) embryos presenting an unclosed wound approximately 16 hr postwounding. Some mutations are ‘‘unscorable’’ due to gross morphological defects either at the wounding stage (15/16) or at the scoring stage (early first instar larvae). (B) Graphic representation of wild- type phenotype (OrR) and the three strongest phenotypes observed. (C–F) wounds in first instar larvae in OrR (C), Jra1 (D), scb5J38 (E), and Rho11B (F). previously implicated in embryonic wound healing similar, each process is distinct in some aspects. In this (Wood et al. 2002). respect, it is worth mentioning that during DC there is We scored wound healing phenotypes as percentage never an actual ‘‘hole’’ to close as the epithelial cells of unclosed wounds. The negative control OrR displayed actually remain in contact with an extraembryonic 5% of phenotype in our assay while the analyzed DC tissue, the amnioserosa (AS), throughout the closure mutants’ phenotype ranged from 2% (shn1) to 74% process. It is now well established that AS cells have an ( Jra1) (Figure 2). The JNK signaling cascade had been active role during DC and it is the fine orchestration of previously demonstrated to be involved in wound the epithelial and AS tissue contributions that ensures healing of larval and adult fly tissues (Ramet et al. successful closure (Kiehart et al. 2000; Hutson et al. 2002; Galko and Krasnow 2004; Bosch et al. 2005). To 2003; Scuderi and Letsou 2005). our knowledge, the Jra1 (null mutation in DJun) mutant Taken together, these pilot results demonstrate both phenotype observed here (74% open wounds) is the the effectiveness and the sensitivity of the wounding first report of the importance of the JNK cascade in fly assay, allowing us to conclude that we developed a embryonic epithelial wound healing. To define a cutoff simple and robust method that can be used in a large- threshold for wound healing defects, we performed a scale genetic screen. Next, we performed a wound statistical analysis on the basis of the maximum likeli- healing screen using a series of largely uncharacterized, hood ratio test (LRT). By comparing all mutants in yet molecularly mapped mutant lines from the Exelixis Figure 2 with OrR we conclude that any line with a stock collection (Thibault et al. 2004). phenotype .30% would be statistically different from Wound healing screen: The Exelixis transposon in- the wild-type situation (5% of open wounds), with a sertion collection is composed of four transposon types: P-value of ,0.001 (data not shown). Therefore, we three piggyBac-based transposons and one P-element- observe a clear wound healing phenotype in Jra1 and based transposon. A subset of this collection likely to scb5J38 (74 and 53%, respectively). The Rho11B phenotype disrupt gene function is present at the Bloomington is not above the threshold but is still significantly Stock Center. We tested all chromosome 2 and 3 lethal different from the control strain (P , 0.005), while all lines in this subset and a few additional viables, for a the other genes tested had either no phenotype or had total of 655 insertional mutant lines (see supporting severe patterning defects at the embryonic stage at information, Table S1). which wounds were made, thus making scoring impos- Of the 655 lines analyzed, we observed 30 (4.6% of the sible (Figure 2). total lines screened) that presented a wound closure The fact that not all tested DC mutants have a wound defect (more than 75 embryos analyzed for each line, healing phenotype suggests that albeit mechanistically Table 1). The total number of mutants with wound Drosophila Wound Healing Screen 133

TABLE 1 Insertional mutants with wound healing phenotypes

Allele tested Molecular function Cellular processes Phenotype (%) aralar1 f05196 Calcium binding, transmembrane Mitochondrial transport 33 (n ¼ 283) transporter arc-p20 e00819 Cytoskeleton component Cell movement, cell shape 43 (n ¼ 189) changes atg2 EP3697 Unknown Autophagy 30 (n ¼ 86) dEaf EP2475 Transcription elongation Stress response 31 (n ¼ 97) dUtx f01321 Chromatin remodeling Gene Silencing 47 (n ¼ 76) glo f02674 Splicing Oogenesis 60 (n ¼ 221) grp e00087 Serine/threonine kinase Cell cycle 44 (n ¼ 171) gs1-like f02438 Glutamate-ammonia ligase Metabolic processes 35 (n ¼ 162) kst d11183 Cytoskeleton component Membrane structure, cell 50 (n ¼ 362) polarity, scaffolding Pc f01890 Chromatin remodeling Gene Silencing 55 (n ¼ 311) Ser12 f03416 Serine protease Proteolysis 44 (n ¼ 212) Stam e00677 JAK pathway signal transduction JAK-STAT signaling 43 (n ¼ 183) adaptor CG2813 f05607 Unknown Unknown 80 (n ¼ 142) CG31805 f07750 Unknown Unknown 33 (n ¼ 114) CG4389 f00822 Long-chain-3 hydroxyacyl- CoA Fatty acid b-oxidation 39 (n ¼167) dehydrogenase CG5198 c07150 Unknown Immune response 49 (n ¼ 147) CG6005 f017117 Unknown Unknown 38 (n ¼ 100) CG6750 e02662 Unknown Unknown 37 (n ¼ 212) CG7627 f01338 Transmembrane transporter Xenobiotic transporter 31 (n ¼ 282) CG9249 f00877 Hexaprenyldihydroxybenzoate Ubiquinone metabolism 51 (n ¼ 182) methyltransferase activity CG10217 f03018 Unknown Unknown 95 (n ¼ 205) CG11089 e03217 IMP cyclohydrolase activity Purine metabolism 35 (n ¼ 130) CG12913 c04874 Acetylgalactosaminyl transferase Chondroitin sulfate biosynthesis 38 (n ¼ 247) CG15170 f06529 Unknown Unknown 43 (n ¼ 75) CG16833 e01119 Tubulin-tyrosine ligase process Protein metabolism 36 (n ¼ 84) CG30010 f01531 Unknown Unknown 42 (n ¼ 104) CG3294 f02075 Splicing Translation regulation 33 (n ¼ 221) CG33123 c03210 Leucine-tRNA ligase tRNA aminoacylation for 48 (n ¼ 280) protein translation Phenotype refers to percentage of total (n) embryos presenting an unclosed wound 16 hr postwounding. Molecular function and cellular processes modified from FlyBase (Tweedie et al. 2009) and additional references mentioned in the text. healing defects is 28 since for two of the lines, Neu3c01955 cable as visualized by phalloidin staining (data not and side f00677, the original insertion location was not shown), suggesting that these mutants must be affecting confirmed (see materials and methods). A number of a process downstream of the initial rapid response of the these mutant alleles were recombined with fluorescent wound by the proximal epithelial cells. markers to allow for observation of tissue morphogen- A karst mutant with wound healing defects: One of esis and wound closure dynamics at the cellular level. In the isolated wound healing mutants is a previously Figure 3, stills taken every 30 min from Jra1, CG2813f05607, unstudied allele of karst (50% open wounds, n ¼ 362, CG5198c07150, and dUtx f01321 mutations recombined with Table 1). This particular mutant, karstd11183,isaP- ubi-DE-cad-GFP (which marks adherens junctions and element-based transposon insertion in the minus orien- allows for cell boundary visualization), show that all four tation at nucleotide position 5219 of the karst ORF, mutant embryos depicted still have open wounds after falling within exon 8. This exon is common to all four 3 hr, whereas in the control embryo (ubi-DE-cad-GFP) predicted karst transcripts (Wilson et al. 2008); there- only a tiny hole remains. These results confirm that our fore, this insertion is likely to disrupt the function of all screen successfully uncovered mutants that display possible gene isoforms. wound healing phenotypes in both larger (assay type) The karst locus encodes the Drosophila bHeavy-Spectrin and smaller, more experimentally tractable wounds, as (bH), which is a large F-actin cross-linking protein the ones depicted in Figure 3. Interestingly, wounds specific to epithelial tissues (Thomas and Kiehart made in these mutants appear to assemble an actin 1994), with orthologs in various species including 134 I. Campos et al.

Figure 3.—Wound closure dynamics in control embryos and wound healing candidate mutants. (A) Stills from movies of wounded control embryos (ubi-DE-Cad-GFP), (B) Jra1, (C) CG2813f05607, (D) CG5198 c07150, and (E) dUtx f01321 embryos recombined with ubi-DE-Cad-GFP, taken 30 (i), 60 (ii), 90 (iii), 120 (iv), and 180 (v) minutes postwounding (mpw). After 3 hr, only wounds in control embryos are nearly closed (compare panel Av to Bv, Cv, Dv, and Ev).

Caenorhabditis elegans and humans (InParanoid eukary- Zarnescu and Thomas 1999; Williams et al. 2004; otic ortholog groups). In epithelial cells, bH, together Praitis et al. 2005). with its binding partner a-spectrin make up the apical The insertion present in the karstd11183 mutant is portion of the spectrin-based membrane skeleton located in segment 15 of bH (see Thomas et al. 1997, (SBMS). This structure is a protein meshwork lying for an explanation of the segment nomenclature). This under the plasma membrane and links the cytoskeleton mutation causes a premature stop codon three amino to the plasma membrane, providing resistance to acids downstream of the insertion site. mechanical stress, while at the same time is possibly karstd11183 mutants have weaker wound-induced actin acting as a scaffold for protein/protein interactions (for cables and less cellular protrusions: To study the review see Thomas 2001). Apart from the structural dynamics of wound healing in the karstd11183 mutant, role, the SBMS is important for maintenance/establish- this line was recombined with sGMCA (the actin ment of the Zona adherens, modulation of the apical binding domain of moesin fused with GFP, Kiehart membrane area, as well as apical constriction and other et al. 2000) and time-lapse recordings were analyzed. contractile actin-ring-based cell morphogenesis occur- Upon wounding, the actin cable was consistently weaker ring during cellularization in Drosophila embryos or and appeared fragmented over time (arrows in Figure body elongation in C. elegans embryos (McKeown et al. 4C, i–iii). Cells at the wound margin do not properly 1998; Thomas et al. 1998; Thomas and Williams 1999; elongate toward the center of the wound and many Drosophila Wound Healing Screen 135

igure F 4.—bH is required for proper epithelial dynamics during wound closure and accumulates robustly in a wound edge cable. (A) Schematic representation of wild-type bH high- lighting known functional domains, numbers in- dicate segments as defined in Thomas et al. (1998). Red arrowhead indicates putative level of protein truncation in karstd11183 mutants. (B and C) Stills from movies of wounded control embryos (sGMCA, B) and bH mutant embryos (karstd11183, C) recombined with sGMCA, taken 15 (i), 60 (ii), and 120 (iii) minutes postwounding (mpw). In control embryos (B) actin accumulates rapidly in a wound edge cable and remains robustly concentrated at the wound margin until closure, while bH mutant embryos fail to accumulate a strong wound edge actin cable (compare actin at wound edge with other cell– cell borders in B vs. C) and cable fragments over time (arrows in C, ii and iii). Only sparse and static filopodia/lamellipodia are seen in mutants while many dynamic protrusions are extended from the wound marginal cells in controls (arrowheads). bH mutant wound marginal cells fail to properly elongate toward the wound center and wound proximal cell edges fail to contract; compare highlighted cells in B with mutant cells in C. (D and E) 3D projections of wounded OrR embryos fixed at 60 mpw. bH protein (shown in red by bH-specific antiserum no. 243 staining) [D(i) and E(i)] accumulates strongly at the wound marginal edge of wound bordering cells in a cable, which colocalizes [yellow in merge E(iii)] with filamentous actin [E(ii)] and with armadillo [D(ii)] at the adherens junctions anchor points [colocalization yellow in merge D(iii)]. Aggregates of red staining in the wound corre- spond to cellular debris and phagocytosed material inside hemocytes.

failed to productively contract their wound marginal responses required to produce a polarized cell shape edges (compare artificially colored wound marginal change, such as cell elongation toward the wound and cells in mutant to those in control in Figure 4C, i–iii wound edge contraction. d11183 with 4B, i–iii, respectively). In addition, we observed bH localizes to wound edges: Given that the karst reduced actin-based protrusion activity at the wound mutant has a wound healing phenotype, we examined edge when compared to wild-type (compare arrowheads whether the bH protein is present at the right time and in Figure 4C, i and ii with 4B, i and ii). After 2 hr, the place to play a direct role in wound closure. It was wounds were still open while corresponding wounds already known that bH is expressed in epithelial tissues made to control embryos closed in 1.5 hr (Figure 4C, throughout embryonic development (Thomas and iehart i–iii and 4B, i–iii). These observations suggest that bH K 1994) but we wanted to know whether it has helps to maintain the actomyosin cable while it is an altered expression or localization pattern within contracting and remodeling as the wound closes. The wounded epithelium. Using bH-specific antiserum no. results further suggest that bH facilitates other spatially 243, which recognizes the N-terminal domain of Dro- restricted actin-based dynamics, such as filopodial sophila bH (Thomas and Kiehart 1994), we observed extension, and may serve to connect the intercellular that the protein concentrates strongly at wound edges cues coming from the actin cable to the intracellular and is present not just at the adherens junction level as 136 I. Campos et al. shown by Armadillo staining (Figure 4D, i–iii), but in a shown). This observation suggests that, in rapidly cable-like pattern that seems to coincide, at least healing epithelial wounds, the JNK pathway is not partially, with the actin cable (Figure 4E, i–iii). This activated to high enough levels to trigger auto- observation is especially interesting because, to our inhibition. knowledge, the only endogenous proteins previously The a-integrin scab was never before implicated in described to localize to the wound edge in a cable-like embryonic wound healing, but this mutant’s phenotype manner are actin and myosin. comes as no great surprise. The first scab mutation was Taken together with the above mutant phenotypes, isolated due to its abnormal larval cuticle patterning these observations suggest that wild-type bH functions (Nusslein-Volhard et al. 1984). The scab gene encodes locally to properly form and/or maintain the intercel- for Drosophila a-PS3 integrin, which is zygotically lular actomyosin cable while it is contracting and expressed in embryonic tissues undergoing invagina- remodeling during wound closure. bH function is also tion, tissue movement, and morphogenesis (Stark et al. required for wild-type intracellular responses such as 1997). Integrin proteins are involved in cell–matrix wound marginal cell edge constriction as well as interactions and a-PS3 integrin regulation, in particu- polarized extension of the wound edge cells toward lar, mediates zipping of opposing epithelial sheets the wound center. Our results further suggest that bH during DC (Homsy et al. 2006). Similarly, our observa- can facilitate other spatially restricted actin-based tion of a wound defect in scb5J38 mutants is consistent events, such as wound edge filopodia dynamics. with a role for a-PS3 integrin in zipping of opposing epithelial cells during the healing process. A previous study using confocal video microscopy has shown that Rho11B mutants take twice as long to close an DISCUSSION epithelial wound when compared to wild type (Wood Wound assay and pilot screen: Using previously et al. 2002). Rho1 was confirmed in our assay to be described DC or wound healing mutants we performed important for wound healing, although with a weaker a pilot screen to validate our embryonic wounding phenotype (22% of embryos had unclosed holes). This strategy. The fact that we identified a member of the result shows nonetheless that our assay can be sensitive DJNK pathway ( Jra/DJun) in our assay is in accordance enough to pick up a ‘‘weak’’ wound healing mutant such with other reports that implicate this pathway in wound as Rho11B, which is still able to heal wounds albeit slower healing. Specifically, two mutations in components of than wild type. the DJNK pathway, bsk/DJNK and kay/DFos, were pre- Transposon screen: The genes identified in the viously shown to have defects in fly larval and adult screen represent a variety of functions indicating that wound closure, respectively (Ramet et al. 2002; Galko wound healing is a complex mechanism that requires and Krasnow 2004). In addition, Mace et al. (2005) the participation of many cellular processes. A large described a reporter construct that requires consensus class of the candidate mutants are involved in several binding sites for the JUN/FOS complex to be activated aspects of gene expression, including factors that upon wounding. Interestingly, the authors still observed regulate chromatin remodeling (dUtx and Pc), elonga- reporter activation in Jra mutants, which suggests that tion (dEaf), splicing (Glo and CG3294), and translation additional signaling pathways are involved in wound (CG33123)(Zink and Paro 1989; Schneider et al. 2004; closure (Mace et al. 2005). Smith et al. 2008; Kalifa et al. 2009; Tweedie et al. An apparent discrepancy arose when our assay re- 2009). These factors are likely needed during wound vealed a phenotype with Jra but not with puc mutants, healing for the induction of a repair transcriptome another component of the same signaling pathway. This (Cooper et al. 2005; Roy et al. 2008; Stramer et al. result might be explained by the fact that Jra and puc 2008). Interestingly, JNK signaling-dependent Pc group function in opposite directions in the DJNK signaling (PcG) gene downregulation has been observed during pathway. Puc functions as a pathway repressor, so in a puc imaginal disc regeneration (Lee et al. 2005). In addition, mutant the JNK pathway should be less repressed and a recent study revealed that PcG methylases are down- we could expect to have an opposite effect to a Jra regulated during wound healing, while counteracting mutation. In addition, we note that activation of a puc– demethylases, Utx and Jmjd3, are upregulated (Shaw lacZ reporter has been shown to occur in larvae, wing and Martin 2009). Our results for the Pc and Utx imaginal discs, and adult wounds that take 18–24 hr to mutants are consistent with these studies and highlight close, but it is only robustly detectable 4–6 hr post- the importance of epigenetic reprogramming in the puncture (Ramet et al. 2002; Galko and Krasnow repair process. 2004; Bosch et al. 2005). Embryonic wounds are faster Some of the genes such as arc-p20 and karst probably to heal, and even after inflicting a large laser wound have a more direct role in the cell shape changes that on stage 14/15 embryos, we failed to detect activation drive the tissue morphogenetic movements during of the puc–lacZ reporter (assessed in open wounds epithelial repair. The gene product of arc-p20 is a 3 hr postwounding by immunofluorescence; data not component of Arp2/3, a complex that controls the Drosophila Wound Healing Screen 137 formation of actin filaments, and karst encodes a that could lead to new paradigms. One hypothesis is component of the spectrin membrane cytoskeleton that Grapes is involved in tension sensing, as it is in the described in detail below (Thomas and Kiehart 1994; spindle checkpoint, or may uncover a cellular repair Kunda et al. 2003; Borghese et al. 2006). Also related to process that could help damaged cells ‘‘decide’’ to morphogenesis, CG12913 encodes an enzyme involved either die by apoptosis or participate in the repair in the synthesis of chondroitin sulfate (Tweedie et al. process. 2009), which is usually found attached to proteins as The remaining genes with a putative function repre- part of a proteoglycan, suggesting a predictable contri- sent a wide range of general metabolic processes bution of the extracellular matrix in the tissue move- (aralar1, gs1-like, CG4389, CG9249, CG11089, and ments necessary for wound healing. CG16833), suggesting that healing the epithelium is a The epithelium is the first line of defense of the highly demanding process (Soehnge et al. 1997; Del organism against pathogens and tissue integrity. It Arco et al. 2000; Tweedie et al. 2009). would thus seem plausible that genes involved in innate Finally, we have also selected a significant number of immunity could be identified with our screening pro- genes that have not yet been studied and do not contain tocol. Indeed, two of the genes (Ser12 and CG5198) identifiable protein domains (CG2813, CG31805, seem to point to the involvement of the immune CG6005, CG6750, CG10217, CG15170, and CG30010). response in the healing of the laser-induced wounds. At the moment it is not possible to predict the role that Ser12 is a member of the serine protease family, a class of these genes may play, but further study may help to proteins that has been shown to play a role in innate identify novel wound healing regulatory mechanisms. immunity (De Gregorio et al. 2001; Ross et al. 2003). Possible role for bH in wound healing: One of the The CG5198 gene has no described function in Dro- mutants identified in our transposon screen was kstd11183, sophila so far, but its homolog, CD2-binding protein 2, is an insertion in the bH-spectrin locus. This mutation is involved in T lymphocyte activation and pre-RNA likely producing a truncated protein terminating three splicing (Kofler et al. 2004; Heinze et al. 2007). amino acids into the P-element insertion (Figure 4A). Another candidate that might represent a link to Other mutations identified in nearby segments 14 immunity is Atg2, a gene important for the regulation (kst14.1, kst2) and 16 (kst1) lead to the production of a of autophagy, a process by which cells degrade cytoplas- detectable truncated protein (Medina et al. 2002) so it is mic components in response to starvation. In Drosoph- likely that karstd11183 mutation also gives rise to a ila, autophagy has been linked to the control of cell truncated protein. These mutant forms of bH lack growth, cell death, and, recently, to the innate immune approximately half of the wild-type protein, including response mechanism against vesicular stomatitis virus a COOH-terminal PH domain region, which is involved and listeria infection (Scott et al. 2004; Yano et al. 2008; in targeting the protein to the membrane (Medina et al. Shelly et al. 2009). 2002), thus producing a potential dominant negative d11183 Isolation of an insertion in the stam gene points to the form of bH. However, the karst mutant should still involvement of the JAK-STAT signaling cascade in this have maternally loaded wild-type protein, as previous regenerative process (Mesilaty-Gross et al. 1999). studies describe a complete absence of maternal protein Interestingly, stam has been shown to be involved in only by the third instar larval stage (Thomas et al. 1998). Drosophila tracheal cell migration and is upregulated This maternal contribution is likely the main reason that following Drosophila larvae infection by Pseudomonas this mutant, as well as the other mutants isolated in our entomophila (Vodovar et al. 2005; Chanut-Delalande screen, does not have a fully penetrant wound healing et al. 2007). phenotype. One candidate could be involved in the uptake or We show for the first time that bH-spectrin localizes to export of some important wound signal (CG7627)as the actomyosin purse string, a supracellular contractile this gene encodes for a multidrug resistant protein cable that forms rapidly upon wound induction. Live (MRP), part of the ABC transporter superfamily, in- imaging has demonstrated that actin and myosin can volved in drug exclusion properties of the Drosophila accumulate in this cable structure within minutes after blood–brain barrier (Tarnay et al. 2004; Mayer et al. wounding (Wood et al. 2002). Unfortunately, due to the 2009). size of the bH gene (.13 kb) cloning and tagging it for The kinase encoded by grapes is the Drosophila live imaging is not possible using standard methods, but homolog of human Check1 (Chk1) involved in the our experiments in fixed tissue tell us that bH can DNA damage and mitotic spindle checkpoints (Fogarty accumulate very rapidly in this cable structure. We have et al. 1997; Furnari et al. 1997; Zachos et al. 2007). To observed bH accumulation at the earliest time point our knowledge, all the Chk1 literature has focused on technically feasible, 15 min postwounding (data not its role during the cell cycle. However, the Drosophila shown). These observations are consistent with previous late embryonic epithelium is a quiescent tissue, even studies, also in fixed tissue, demonstrating rapid after wounding (data not shown). Understanding changes in bH localization during the process of Grapes function in this context is a challenging task cellularization in Drosophila embryos (Thomas and 138 I. Campos et al.

Williams 1999). Taken together, it is clear that at least data and the published work discussed above, introduc the bH component of the membrane skeleton is not just the intriguing possibility that bH could be serving as a a static structural scaffold as the name implies, but link between wound edge dynamics and the coordi- rather a dynamic protein capable of responding to or nated cell shape changes usually observed in wild-type directing changes in cellular dynamics. Our studies wound edge cells. The combination of the proposed suggest that polarized redistribution of bH exerts an ability of bH to modulate the apical membrane area as essential function to facilitate actin-based cellular re- well as cross-link actin and act as an apical membrane- sponses, such as cable accumulation/maintenance and wide scaffold for other interactions, makes bH a good wound edge filopodia dynamics, which are necessary to candidate to provide the physical link that would properly close a wound. coordinate tissuewide actions, such as supracellular bH as a link between cell membranes and contractile actin cable contraction, with the individual cellular rings: bH has been previously observed in association responses, such as cell shape change and polarized with actin ‘‘rings’’ during development of Drosophila filopodia activity. homas and C. elegans (reviewed in T 2001). Arguably, C. We thank Vı´tor Barbosa, Luı´s Teixeira, and Soren Prag for critically elegans provides an example of actin ring function most reading the manuscript and all the lab members for helpful discussions. analogous to our wound edge purse string. During the We thank Graham Thomas for the generous gift of bH-specific final stages of C. elegans development, cortical arrays of antiserum no. 243 and Helena Igle´sias for help with the statistical actin in the outer epithelial cells, the hypodermis, analysis. This work was supported by Fundacxaõ para a Cieˆncia e a Tecnologia (I.C. SFRH/BPD/17729/2004; J.A.G. SFRH/BD/21895/ dramatically reorganize to form parallel apically local- 2005, PTDC/BIA-BCM/65872/2006, A.C.S. SFRH/BPD/24976/2005), ized bundles of circumferencial supracellular actin European Union Framework Programs (LSHM-CT-2003-504468, ERC- rings (McKeown et al. 1998; Praitis et al. 2005). In this 2007-StG), and Human Frontiers Science Program (RGP21/2007). system, sma1, the C. elegans ortholog of bH, also localizes apically to these actin rings. In sma1 mutants the rings fail to productively contract and begin to disorganize, LITERATURE CITED losing connection to the cell membranes. An additional Arora, K., H. Dai,S.G.Kazuko,J.Jamal,M.B.O’Connor et al., phenotype observed in these mutants is the inability of 1995 The Drosophila schnurri gene acts in the Dpp/TGF beta cells to change their shape, a process normally ‘‘di- signaling pathway and encodes a transcription factor homolo- gous to the human MBP family. Cell 81: 781–790. rected’’ by these contractile rings, the end result being a Borghese,L.,G.Fletcher,J.Mathieu,A.Atzberger,W.C. short worm, a phenotype we see as functionally analo- Eades et al., 2006 Systematic analysis of the transcriptional gous to an unclosed wound in our system. switch inducing migration of border cells. Dev. Cell 10: 497– 508. In Drosophila, bH has been previously implicated in Bosch, M., F. Serras,E.Martin-Blanco and J. Baguna, 2005 JNK modulating cell shape changes during apical constric- signaling pathway required for wound healing in regenerating tion of follicle cells (a process also involving actin rings) Drosophila wing imaginal discs. Dev. Biol. 280: 73–86. Chanut-Delalande, H., A. C. Jung,L.Lin,M.M.Baer,A.Bilstein and has been proposed to function as a link between et al., 2007 A genetic mosaic analysis with a repressible cell cross-linked actin networks/rings and the cell mem- marker screen to identify genes involved in tracheal cell migra- brane (Thomas 2001). Further studies revealed that the tion during Drosophila air sac morphogenesis. Genetics 176: 2177–2187. C-terminal domain of bH has the ability to directly Ciapponi, L., D. B. Jackson,M.Mlodzik and D. Bohmann, modulate the apical membrane area by regulating 2001 Drosophila Fos mediates ERK and JNK signals via distinct endocytosis (Williams et al. 2004), adding one more phosphorylation sites. Genes Dev. 15: 1540–1553. ooper ohnson urslem artin b C , L., C. J ,F.B and P. M , 2005 Wound tantalizing piece of evidence pointing to the fact that H healing and inflammation genes revealed by array analysis of could be a major player in cell shape changes, not only ‘macrophageless’ PU.1 null mice. Genome Biol. 6: R5. as a structural link but also by directly modulating the Davidson, L. A., A. M. Ezin and R. Keller, 2002 Embryonic wound healing by apical contraction and ingression in Xenopus laevis. membrane area in response to cytoskeletal clues (or vice Cell Motil. Cytoskeleton 53: 163–176. versa). De Gregorio, E., P. T. Spellman,G.M.Rubin and B. Lemaitre, Although we know from previous studies that the 2001 Genome-wide analysis of the Drosophila immune re- actin cable is not absolutely required for wound closure sponse by using oligonucleotide microarrays. Proc. Natl. Acad. Sci. USA 98: 12590–12595. (Wood et al. 2002), the process takes much longer Del Arco, A., M. Agudo and J. Satrustegui, without one. In Rho1 mutant embryos, cells lacking a 2000 Characterization of a second member of the subfamily cable are able to pull the wound closed using filopodia of calcium-binding mitochondrial carriers expressed in human ood non-excitable tissues. Biochem. J. 345(Pt 3): 725–732. (W et al. 2002). The filopodial defect observed in Fernandez, R., F. Takahashi,Z.Liu,R.Steward,D.Stein et al., karst mutants, adds another line of evidence to the 2000 The Drosophila shark tyrosine kinase is required for em- absolute requirement of these structures for wound bryonic dorsal closure. Genes Dev. 14: 604–614. Fogarty, P., S. D. Campbell,R.Abu-Shumays,B.S.Phalle,K.R.Yu closure. In addition to the reduced actin cable accumu- et al., 1997 The Drosophila grapes gene is related to checkpoint lation and filopodial dynamics in karst mutants (which gene chk1/rad27 and is required for late syncytial division fidel- would lack the C-terminal domain responsible for ity. Curr. Biol. 7: 418–426. Frank, L. H., and C. Rushlow, 1996 A group of genes required for membrane modulation), we also see a lack of cell shape maintenance of the amnioserosa tissue in Drosophila. Develop- change in the wound edge cells. Taken together, these ment 122: 1343–1352. Drosophila Wound Healing Screen 139

Furnari, B., N. Rhind and P. Russell, 1997 Cdc25 mitotic inducer Mesilaty-Gross, S., A. R.Meich,B otro and R. Wides, 1999 The targeted by chk1 DNA damage checkpoint kinase. Science 277: Drosophila STAM gene homolog is in a tight gene cluster, and its 1495–1497. expression correlates to that of the adjacent gene ial. Gene 231: Galko, M. J., and M. A. Krasnow, 2004 Cellular and genetic anal- 173–186. ysis of wound healing in Drosophila larvae. PLoS Biol. 2: E239. Nellen, D., M. Affolter and K. Basler, 1994 Receptor serine/ Garcia-Fernandez B.,I.Campos,J.Geiger,A.C.Santos and A. Jacinto, threonine kinases implicated in the control of Drosophila body 2009 Epithelial resealing. Int. J. Dev. Biol. 53: 1549–1556. pattern by decapentaplegic. Cell 78: 225–237. Heinze, M., M. Kofler and C. Freund, 2007 Investigating the func- Nusslein-Volhard, C., E. Wieschaus and H. Kluding, tional role of CD2BP2 in T cells. Int. Immunol. 19: 1313–1318. 1984 Mutations affecting the pattern of the larval cuticle in Homsy, J. G., H. Jasper,X.G.Peralta,H.Wu,D.P.Kiehart et al., Drosophila melanogaster. I. Zygotic loci on the second chromo- 2006 JNK signaling coordinates integrin and actin functions some. Rouxs Arch. Dev. Biol. 193: 267–282. during Drosophila embryogenesis. Dev. Dyn. 235: 427–434. Oda, H., and S. Tsukita, 2001 Real-time imaging of cell-cell adhe- Hutson, M. S., Y. Tokutake,M.S.Chang,J.W.Bloor,S.Venakides rens junctions reveals that Drosophila mesoderm invagination et al., 2003 Forces for morphogenesis investigated with laser mi- begins with two phases of apical constriction of cells. J. Cell crosurgery and quantitative modeling. Science 300: 145–149. Sci. 114: 493–501. Jacinto, A., W. Wood,T.Balayo,M.Turmaine,A.Martinez-Arias Pearson, J. C., M. T. Juarez,M.Kim and W. McGinnis, et al., 2000 Dynamic actin-based epithelial adhesion and cell 2009 Multiple transcription factor codes activate epidermal matching during Drosophila dorsal closure. Curr. Biol. 10: wound-response genes in Drosophila. Proc. Natl. Acad. Sci. 1420–1426. USA 106: 2224–2229. raitis iccone ustin Jacinto, A., A. Martinez-Arias and P. Martin, 2001 Mechanisms P , V., E. C and J. A , 2005 SMA-1 spectrin has es- of epithelial fusion and repair. Nat. Cell. Biol. 3: E117–E123. sential roles in epithelial cell sheet morphogenesis in C. elegans. Kalifa, Y., S. T. Armenti and E. R. Gavis, 2009 Glorund interac- Dev. Biol. 283: 157–170. rice avenye um reitkreutz tions in the regulation of gurken and oskar mRNAs. Dev. Biol. P , J. V., E. D. S ,D.L and A. B , 326: 68–74. 1997 Dominant enhancers of Egfr in Drosophila melanogaster: ge- Kiehart, D. P., C. G. Galbraith,K.A.Edwards,W.L.Rickoll and netic links between the Notch and Egfr signaling pathways. Ge- R. A. Montague, 2000 Multiple forces contribute to cell sheet netics 147: 1139–1153. amet anot achary anfruelli morphogenesis for dorsal closure in Drosophila. J. Cell Biol. 149: R , M., R. L ,D.Z and P. M , 2002 JNK 471–490. signaling pathway is required for efficient wound healing in Dro- ockel eitlinger taszewski lodzik sophila. Dev. Biol. 241: 145–156. K , L., J. Z ,L.M.S ,M.M and D. ing artinez rias Bohmann, 1997 Drosophila development: redundant and non- R , J. M., and A. M A , 1993 puckered, a gene in- redundant functions and regulation by two MAPK signal trans- volved in position-specific cell differentiation in the dorsal epidermis of the Drosophila larva. Dev. (Suppl.): 251–259. duction pathways. Genes Dev. 11: 1748–1758. oss iang anost ang Kofler, M., K. Heuer,T.Zech and C. Freund, 2004 Recognition R , J., H. J ,M.R.K and Y. W , 2003 Serine pro- sequences for the GYF domain reveal a possible spliceosomal teases and their homologs in the Drosophila melanogaster genome: an initial analysis of sequence conservation and function of CD2BP2. J. Biol. Chem. 279: 28292–28297. Kunda, P., G. Craig,V.Dominguez and B. Baum, 2003 Abi, Sra1, phylogenetic relationships. Gene 304: 117–131. Roy, S., S. Khanna,C.Rink,S.Biswas and C. K. Sen, and Kette control the stability and localization of SCAR/WAVE 2008 Characterization of the acute temporal changes in exci- to regulate the formation of actin-based protrusions. Curr. Biol. sional murine cutaneous wound inflammation by screening of 13: 1867–1875. the wound-edge transcriptome. Physiol. Genomics 34: 162–184. Lee, N., C. Maurange,L.Ringrose and R. Paro, 2005 Suppression Schneider, C., C. L. Will,J.Brosius,M.J.Frilander and R. of Polycomb group proteins by JNK signalling induces transde- Luhrmann, 2004 Identification of an evolutionarily divergent termination in Drosophila imaginal discs. Nature 438: 234–237. U11 small nuclear ribonucleoprotein particle in Drosophila. Lekven, A. C., U. Tepass,M.Keshmeshian and V. Hartenstein, Proc. Natl. Acad. Sci. USA 101: 9584–9589. 1998 faint sausage encodes a novel extracellular protein of Schock, F., and N. Perrimon, 2003 Retraction of the Drosophila the immunoglobulin superfamily required for cell migration germ band requires cell-matrix interaction. Genes Dev. 17: and the establishment of normal axonal pathways in the Dro- 597–602. sophila nervous system. Development 125: 2747–2758. cott chuldiner eufeld i ustafson rown anks ason rbeit S , R. C., O. S and T. P. N , 2004 Role and reg- L , G., C. G -B ,S.K.H ,K.N ,J.M.A ulation of starvation-induced autophagy in the Drosophila fat et al., 2003 c-Jun is essential for organization of the epidermal body. Dev. Cell 7: 167–178. leading edge. Dev. Cell 4: 865–877. Scuderi, A., and A. Letsou, 2005 Amnioserosa is required for dor- ace earson c innis M , K. A., J. C. P and W. M G , 2005 An epidermal sal closure in Drosophila. Dev. Dyn. 232: 791–800. barrier wound repair pathway in Drosophila is mediated by Shaw, T., and P. Martin, 2009 Epigenetic reprogramming during grainy head. Science 308: 381–385. wound healing: loss of polycomb-mediated silencing may enable agie arkhurst M , C. R., and S. M. P , 2005 Rho1 regulates signaling upregulation of repair genes. EMBO Rep. 10: 881–886. events required for proper Drosophila embryonic development. Shelly, S., N. Lukinova,S.Bambina,A.Berman and S. Cherry, Dev. Biol. 278: 144–154. 2009 Autophagy is an essential component of Drosophila im- artin ewis M , P., and J. L , 1992 Actin cables and epidermal move- munity against vesicular stomatitis virus. Immunity 30: 588–598. ment in embryonic wound healing. Nature 360: 179–183. Smith, E. R., B. Winter,J.C.Eissenberg and A. Shilatifard, artin arkhurst M , P., and S. M. P , 2004 Parallels between tissue re- 2008 Regulation of the transcriptional activity of poised RNA pair and embryo morphogenesis. Development 131: 3021–3034. polymerase II by the elongation factor ELL. Proc. Natl. Acad. ayer ayer hinn insonneault roetz M , F., N. M ,L.C ,R.L.P ,D.K et al., Sci. USA 105: 8575–8579. 2009 Evolutionary conservation of vertebrate blood-brain bar- Soehnge, H., X. Huang,M.Becker,D.Conover and M. Stern, rier chemoprotective mechanisms in Drosophila. J. Neurosci. 1997 Cloning and sequencing of ribosomal protein L27a and 29: 3538–3550. a gene similar to human GS1 in Drosophila. Gene 185: 257–263. McCluskey, J., J. Hopkinson-Woolley,B.Luke and P. Martin, Spradling, A. C., D. Stern,A.Beaton,E.J.Rhem,T.Laverty et al., 1993 A study of wound healing in the E11.5 mouse embryo 1999 The Berkeley Drosophila Genome Project gene disrup- by light and electron microscopy. Tissue Cell 25: 173–181. tion project: Single P-element insertions mutating 25% of vital McKeown, C., V. Praitis and J. Austin, 1998 sma-1 encodes a be- Drosophila genes. Genetics 153: 135–177. taH-spectrin homolog required for Caenorhabditis elegans mor- Stark, K. A., G. H. Yee,C.E.Roote,E.L.Williams,S.Zusman et al., phogenesis. Development 125: 2087–2098. 1997 A novel alpha integrin subunit associates with betaPS and Medina, E., J. Williams,E.Klipfell,D.Zarnescu,G.Thomas et al., functions in tissue morphogenesis and movement during Dro- 2002 Crumbs interacts with moesin and beta(Heavy)-spectrin sophila development. Development 124: 4583–4594. in the apical membrane skeleton of Drosophila. J. Cell Biol. Stramer, B., M. Winfield,T.Shaw,T.H.Millard,S.Woolner 158: 941–951. et al., 2008 Gene induction following wounding of wild-type ver- 140 I. Campos et al.

sus macrophage-deficient Drosophila embryos. EMBO Rep. 9: Vodovar, N., M. Vinals,P.Liehl,A.Basset,J.Degrouard et al., 465–471. 2005 Drosophila host defense after oral infection by an ento- Tarnay, J. N., F. Szeri,A.Ilias,T.Annilo,C.Sung et al., 2004 The mopathogenic Pseudomonas species. Proc. Natl. Acad. Sci. dMRP/CG6214 gene of Drosophila is evolutionarily and func- USA 102: 11414–11419. tionally related to the human multidrug resistance-associated Wang,S.,V.Tsarouhas,N.Xylourgidis,N.Sabri,K.Tiklova protein family. Insect Mol. Biol. 13: 539–548. et al., 2009 The tyrosine kinase Stitcher activates Grainy head Thibault, S. T., M. A. Singer,W.Y.Miyazaki,B.Milash,N.A. and epidermal wound healing in Drosophila. Nat. Cell Biol. 11: Dompe et al., 2004 A complementary transposon tool kit for 890–895. Drosophila melanogaster using P and piggyBac. Nat. Genet. Williams, J. A., B. MacIver,E.A.Klipfell and G. H. Thomas, 36: 283–287. 2004 The C-terminal domain of Drosophila (beta) heavy-spec- Thomas, G. H., 2001 Spectrin: the ghost in the machine. Bioessays trin exhibits autonomous membrane association and modulates 23: 152–160. membrane area. J. Cell Sci. 117: 771–782. Thomas, G. H., and D. P. Kiehart, 1994 Beta heavy-spectrin has a Wilson,R.J.,J.L.Goodman,V.B.Strelets and FlyBase Consortium, restricted tissue and subcellular distribution during Drosophila 2008 FlyBase: integration and improvements to query tools. embryogenesis. Development 120: 2039–2050. Nucleic Acids Res. 36: D588–D593. Thomas, G. H., and J. A. Williams, 1999 Dynamic rearrangement Wood, W., A. Jacinto,R.Grose,S.Woolner,J.Gale et al., of the spectrin membrane skeleton during the generation of ep- 2002 Wound healing recapitulates morphogenesis in Drosoph- ithelial polarity in Drosophila. J. Cell Sci. 112(Pt 17): 2843–2852. ila embryos. Nat. Cell Biol. 4: 907–912. Thomas, G. H., E. C. Newbern,C.C.Korte,M.A.Bales,S.V.Muse Wu, Y., A. R. Brock,Y.Wang,K.Fujitani,R.Ueda et al., 2009 A et al., 1997 Intragenic duplication and divergence in the spec- blood-borne PDGF/VEGF-like ligand initiates wound-induced trin superfamily of proteins. Mol. Biol. Evol. 14: 1285–1295. epidermal cell migration in Drosophila larvae. Curr. Biol. 19: Thomas,G.H.,D.C.Zarnescu,A.E.Juedes,M.A.Bales,A.Londergan 1473–1477. et al., 1998 Drosophila betaHeavy-spectrin is essential for devel- Yano, T., S. Mita,H.Ohmori,Y.Oshima,Y.Fujimoto et al., opment and contributes to specific cell fates in the eye. Development 2008 Autophagic control of listeria through intracellular innate 125: 2125–2134. immune recognition in drosophila. Nat. Immunol. 9: 908–916. Ting,S.B.,T.Wilanowski,A.Auden,M.Hall,A.K.Voss et al., Young, P. E., A. M. Richman,A.S.Ketchum and D. P. Kiehart, 2003 Inositol- and folate-resistant neural tube defects in mice lack- 1993 Morphogenesis in Drosophila requires nonmuscle myosin ing the epithelial-specific factor Grhl-3. Nat. Med. 9: 1513–1519. heavy chain function. Genes Dev. 7: 29–41. Ting, S. B., J. Caddy,N.Hislop,T.Wilanowski,A.Auden et al., Zachos, G., E. J. Black,M.Walker,M.T.Scott,P.Vagnarelli 2005a A homolog of Drosophila grainy head is essential for epi- et al., 2007 Chk1 is required for spindle checkpoint function. dermal integrity in mice. Science 308: 411–413. Dev. Cell 12: 247–260. Ting, S. B., J. Caddy,T.Wilanowski,A.Auden,J.M.Cunningham Zarnescu, D. C., and G. H. Thomas, 1999 Apical spectrin is essen- et al., 2005b The epidermis of grhl3-null mice displays altered tial for epithelial morphogenesis but not apicobasal polarity in lipid processing and cellular hyperproliferation. Organogenesis Drosophila. J. Cell Biol. 146: 1075–1086. 2: 33–35. Z ink, B., and R. Paro, 1989 In vivo binding pattern of a trans-reg- Tweedie, S., M. Ashburner,K.Falls,P.Leyland,P.McQuilton ulator of homoeotic genes in Drosophila melanogaster. Nature et al., 2009 FlyBase: enhancing Drosophila Gene Ontology an- 337: 468–471. notations. Nucleic Acids Res. 37: 10.1093/nar/gkn1788. Uv, A. E., E. J. Harrison and S. J. Bray, 1997 Tissue-specific splicing and functions of the Drosophila transcription factor Grainy- head. Mol. Cell. Biol. 17: 6727–6735. Communicating editor: D. I. Greenstein

Supporting Information http://www.genetics.org/cgi/content/full/genetics.109.110288/DC1

Genetic Screen in Drosophila melanogaster Uncovers a Novel Set of Genes Required for Embryonic Epithelial Repair

Isabel Campos, Jennifer A. Geiger, Ana Catarina Santos, Vanessa Carlos and Antonio Jacinto

DOI: 10.1534/genetics.109.110288

2 SI I. Campos et al.

TABLE S1

Insertional mutants screened for wound healing phenotypes

Insertional Mutant n= % holes

140upf07279 33 12.1

Aats-metc00449 61 4.9

Aats-proe00080 59 0.0

abod01007 57 1.8

absf01698 30 10.0

Acec00215 62 1.6

Actn3f00971 50 6.0

Akap200EP2254 41 0.0

alf04261 42 11.9

amdf03321 38 10.5

Antpd06610 0 *

Aos1c06048 206 27.7

AP-2f03132 56 3.6

Apc2c03232 48 8.3

aralar1f05196 283 33.2

Arc-p20e00819 189 43.4

Arf51FEP2612 37 0.0

Arp5e02275 49 14.3

Arp87Cc04425 92 22.8

Atg2EP3697 86 30.2

Atg7d06996 0 **

att-ORFAe03144 or att-ORFBe03144 46 15.2

Awhc05541 49 2.0

bchse00833 213 11.7 I. Campos et al. 3 SI

beat-VIc06401 56 8.9

Best1f07188 86 14.0

Brfc07161 66 0.0

Brucee00984 59 16.9

Btk29AEP2167 52 7.7 c(2)MEP2115 83 0.0 c02483 25 0.0 c05413 55 5.5 c05496 51 0.0 c05504b 40 10.0 c07013 26 0.0

Cad96Cad07355 65 3.1

CAP-D2f03381 48 2.1

Cap-D3f02191 182 26.4

Cap-Gc00093 89 18.0

Cap-GEP2346 10 0.0 captf00786 45 8.9 capuf02268 96 21.9

Cct1EP3346 0 * cdc2c03495 31 9.7

Cdk9f05537 57 17.5

Ced-12c06760 48 12.5

CG10137f04546 40 10.0

CG10139c01615 36 2.8

CG10154c02170 57 1.8

CG10168f03105 43 7.0

CG10195f06699 55 0.0 4 SI I. Campos et al.

CG10217f03018 205 95.1

CG10253f02060 94 22.3

CG10289f02530 35 0.0

CG10333d02040 38 21.1

CG10341f07749 50 8.0

CG10343f03911 111 15.3

CG10413EP2164 50 2.0

CG10414e03046 52 3.8

CG10420c06294 110 10.9

CG10561f06260 76 15.8

CG10600d07626 160 29.4

CG10602f04195 or mRpL13f04195 40 27.5

CG10627c04986 67 14.9

CG10628e01892 69 27.5

CG10635c00409 44 13.6

CG10650d00783 90 7.8

CG10754f03161 43 0.0

CG10907e00319 46 21.7

CG11007EP736 55 3.6

CG11030c03484 127 27.6

CG11070EP2597 18 0.0

CG11089e03217 130 35.4

CG11166EP2475 97 30.9

CG11180e03938 67 3.0

CG11188e03057 99 11.1

CG11200d02302 55 10.9

CG11241d06786 62 12.9 I. Campos et al. 5 SI

CG11266f07714 66 13.6

CG11319f06271 40 2.5

CG11419e01070 39 5.1

CG11426d05846 49 4.1

CG11455d08265 47 12.8

CG11486EP3109 55 0.0

CG11490d05023 59 0.0

CG11583c01124 39 0.0

CG11593d06001 52 0.0

CG11836f02631 55 18.2

CG11851c02021 112 27.7

CG11896d05816 61 0.0

CG11927c04401 or mRpS2c04401 133 22.6

CG11984d08881 42 4.8

CG12063f06602 104 20.2

CG12140f05640 100 29.0

CG12267f05999 40 0.0

CG12314f01342 143 23.1

CG1234e01488 58 3.4

CG12413c02868 114 27.2

CG12753f07675 69 0.0

CG12822f02022 48 8.3

CG12901f07056 70 7.1

CG12913c04874 247 37.7

CG12934e03982 90 27.8

CG13018e00535 58 0.0

CG13130EP2238 56 8.9 6 SI I. Campos et al.

CG13131f04310 84 0.0

CG13379e01308 47 8.5

CG13398c02876 49 0.0

CG1344EP2646 54 1.9

CG13466f02655 42 2.4

CG13527f04582 63 23.8

CG13551e03979 63 7.9

CG13594f00918 57 22.8

CG13689e00773 52 9.6

CG13776c06748 88 12.5

CG13793f01503 68 1.5

CG13993EP570 35 5.7

CG14017d05326 51 0.0

CG14023e04200 44 13.6

CG14057e03520 52 0.0

CG14133f05716 59 5.1

CG14275d00140 48 14.6

CG14544f01091 42 2.4

CG14598e01297 67 9.0

CG14655e04480 35 2.9

CG14830e00332 48 4.2

CG14894f04937 43 7.0

CG14955c02957 52 0.0

CG1499c06953 31 16.1

CG15170f06529 75 42.7

CG15173f05160 21 4.8

CG15436f07761 51 2.0 I. Campos et al. 7 SI

CG15443c03956 or GRHRc03956 83 8.4

CG15443e03827 34 2.9

CG15544e03116 101 5.0

CG15625e03818 36 8.3

CG15626f03581 56 12.5

CG15635f08079 98 19.4

CG15695c04218 55 1.8

CG15696c04976 60 8.3

CG15706f06892 or CG15701f06892 48 12.5

CG1600EP398 140 18.6

CG1603f04743 62 3.2

CG1607e00971 108 24.1

CG16786e03819 60 6.7

CG1681f06400 38 7.9

CG16833e01119 84 35.7

CG16865f04739 53 5.7

CG16884c05307 and eygc05307 33 18.2

CG16908e01001 54 3.7

CG16947e00837 50 14.0

CG16979e02981 64 14.1

CG17086e02595 28 10.7

CG17141f03838 84 11.9

CG17202f01979 116 17.2

CG17219e03045 36 0.0

CG17221c00569 61 0.0

CG17350e03687 48 14.6

CG17379f05980 40 17.5 8 SI I. Campos et al.

CG17562f02635 68 13.2

CG17597e00674 110 0.9

CG17612c04955 91 18.7

CG17658EP730 57 7.0

CG17712f05481 63 0.0

CG1776f01512 75 8.0

CG17838c04375 or Takl1c04375 105 21.0

CG17912EP819 52 5.8

CG18131f04301 51 2.0

CG18156f03756 60 0.0

CG18522d03463 62 19.4

CG18539f02127 48 8.3

CG18606e04639 55 12.7

CG18619c02273 51 7.8

CG1887f07156 65 0.0

CG1894f06204 49 4.1

CG1957f00376 25 8.0

CG1962f04787 26 3.8

CG2107f02667 50 0.0

CG2121f00867 53 5.7

CG2493c06582 118 21.2

CG2747c02682 40 12.5

CG2767e02843 103 15.5

CG2813f05607 142 79.6

CG2921e02644 36 2.8

CG2950c00722 55 9.1

CG30010f01531 104 42.3 I. Campos et al. 9 SI

CG30158f06824 65 3.1

CG30290f00839 55 1.8

CG30431e01618 58 8.6

CG30437f04551 42 14.3

CG30497EP2618 106 0.0

CG31004f04955 59 10.2

CG31005c04819 137 22.6

CG31120e00272 61 27.9

CG31121f07249 36 16.7

CG31211e01498 55 5.5

CG31316f01681 51 5.9

CG31337e04105 65 10.8

CG31368e00215 157 29.3

CG31374f07849 or CG17230f07849 48 6.3

CG31600f04602 20 0.0

CG31718f06333 47 0.0

CG31728f02493 53 5.7

CG31731f02254 47 14.9

CG31738e02963 58 17.2

CG31805f07750 114 33.3

CG31851f03572 42 11.9

CG31855e03029 140 27.1

CG31871f02763 34 20.6

CG31878e00164 73 1.4

CG31898e03937 46 0.0

CG31901f00026 64 3.1

CG31902c02416 52 0.0 10 SI I. Campos et al.

CG31908f00433 51 11.8

CG31935c00037 53 3.8

CG31957f05539 43 18.6

CG31960f02028 50 24.0

CG31961f06868 18 22.2

CG31973c04017 47 0.0

CG32085c01612 60 3.3

CG32111f03764 156 15.4

CG32133c04574 56 7.1

CG32176f05870 52 5.8

CG32260c05553 68 10.3

CG32352d11266 58 3.4

CG32373c06299 60 3.3

CG32373f02001 or Ank2f02001 0 *

CG32388f06166 149 18.1

CG32409c03942 63 3.2

CG32418e03237 62 17.7

CG3267e02164 56 7.1

CG3285f03337 65 1.5

CG3294f02075 221 32.6

CG32972e03947 41 14.6

CG33116e02587 48 2.1

CG33123c00827 163 13.5

CG33123c03210 280 47.9

CG33322f00115 61 13.1

CG3335c05958 27 11.1

CG33936f01391 51 5.9 I. Campos et al. 11 SI

CG34124f07566 112 8.0

CG34127f00777 49 0.0

CG34363f03574 57 8.8

CG34366f04377 68 0.0

CG34367f00117 59 6.8

CG34380f05536 42 16.7

CG34406c02114 98 16.3

CG3542EP719 56 17.9

CG3563f00590 56 8.9

CG3590c02781 198 26.8

CG3609e02184 107 29.0

CG3609e02299 48 16.7

CG3645f02260 or CG3345f02260 51 21.6

CG3662e04640 51 3.9

CG3683c06534 44 0.0

CG3700d06151 43 2.3

CG3714EP2611 58 12.1

CG3764f04642 65 3.1

CG3803f04773 50 4.0

CG3983c07050 55 29.1

CG4389f00822 167 38.9

CG4398d00549 61 9.8

CG4484f01710 42 4.8

CG4497f05611 119 22.7

CG4554c02084 85 0.0

CG4594f04335 113 4.4

CG4658f00098 56 3.6 12 SI I. Campos et al.

CG4674d06455 27 3.7

CG4738EP372 50 0.0

CG4757c07133 43 0.0

CG4774c01874 116 14.7

CG4774e01021 76 18.4

CG4836c04238 75 5.3

CG4848e02840 51 2.0

CG4851c02813 58 6.9

CG4933f01978 50 4.0

CG4942f00751 26 0.0

CG4959f02253 24 0.0

CG5003f02616 48 8.3

CG5091f04215 59 3.4

CG5126f00916 53 1.9

CG5147e03412 48 6.3

CG5148c03646 48 0.0

CG5149c00700 61 1.6

CG5149e03983 70 18.6

CG5156c05647 53 0.0

CG5168c04880 38 5.3

CG5181c01211 46 4.3

CG5189e01140 41 7.3

CG5198c07150 147 49.0

CG5276e03505 68 2.9

CG5290f02563 91 28.6

CG5342e01976 64 17.2

CG5384f00779 55 5.5 I. Campos et al. 13 SI

CG5451e03563 71 1.4

CG5451f03090 64 9.4

CG5508f04927 36 2.8

CG5515f02614 93 5.4

CG5525EP682 51 2.0

CG5567f05921 88 10.2

CG5589f06152 53 0.0

CG5602f00902 46 6.5

CG5626e02731 30 10.0

CG5640f01321 76 47.4

CG5645e03479 48 0.0

CG5660f01783 93 6.5

CG5758c01197 112 28.6

CG5758e01537 59 13.6

CG5780EP2315 or EP2315EP2315 46 2.2

CG5807c05043 41 2.4

CG5850c03122 36 5.6

CG5888e02257 or Idgf2e02257 61 6.6

CG5931e03171 62 14.5

CG5970e00083 41 22.0

CG6005f07117 100 38.0

CG6113f07089 67 10.4

CG6126d09805 64 6.3

CG6136e02834 36 2.8

CG6171d07444 43 0.0

CG6180c02598 233 9.0

CG6196f00985 107 10.3 14 SI I. Campos et al.

CG6225c03494 13 0.0

CG6357d03281 60 23.3

CG6393EP2457 57 12.3

CG6568EP2499 54 9.3

CG6608f01677 36 5.6

CG6637f01832 35 8.6

CG6678d10234 59 8.5

CG6686f06314 126 19.0

CG6724e02149 59 0.0

CG6729c04960 43 0.0

CG6739d09967 46 4.3

CG6746EP1118 51 17.6

CG6750e02662 212 37.3

CG6792c06236 60 0.0

CG6856e01028 120 18.3

CG6907d06812 224 27.7

CG6931f00440 52 5.8

CG6951e00910 33 0.0

CG7029c06518 64 12.5

CG7081f01899 59 15.3

CG7191f03241 43 0.0

CG7202f03815 49 2.0

CG7214c01614 72 0.0

CG7263e042 55 18.2

CG7371f02109 75 9.3

CG7394c04985 0 *

CG7532e01905 70 5.7 I. Campos et al. 15 SI

CG7549c05288 31 0.0

CG7627f01338 282 30.9

CG7638e04494 52 1.9

CG7639e02922 16 6.3

CG7755f06581 0 **

CG7806f02044 103 27.2

CG7816f00306 66 1.5

CG7816f02876 50 2.0

CG7818e00875 82 24.4

CG7845c00845 60 8.3

CG7861f00024 61 13.1

CG7870e04276 64 18.8

CG7891e00336 40 5.0

CG7918f01078 40 5.0

CG8064c05886 75 6.7

CG8083EP2412 43 0.0

CG8086f03214 46 0.0

CG8233d11598 105 21.9

CG8270f03412 30 13.3

CG8412f07214 61 9.8

CG8414EP525 59 11.9

CG8419e04179 40 7.5

CG8494e04168 83 27.7

CG8516d10724 33 3.0

CG8552c03991 or CG31607c03991 59 25.4

CG8552f04269 105 23.8

CG8745e00991 58 3.4 16 SI I. Campos et al.

CG8777f03251 67 13.4

CG8861f06147 34 5.9

CG9003d09761 78 1.3

CG9143e00691 54 25.9

CG9162d03190 60 0.0

CG9249f00835 93 7.5

CG9249f00877 182 51.1

CG9264d03356 30 3.3

CG9265d00690 79 8.9

CG9289e02128 72 27.8

CG9293f03884 93 24.7

CG9296f04175 49 0.0

CG9320c06563 51 25.5

CG9510c05706 or CG9510c05706 52 1.9

CG9555f00882 88 15.9

CG9596e01579 46 19.6

CG9603d04921 32 9.4

CG9603e03209 59 8.5

CG9669f07000 68 1.5

CG9778e00450 105 27.6

CG9922f01835 34 2.9

CG9932c00144 60 0.0

CG9987f05494 70 7.1

CheA29af07701 130 5.4

CHORDc02881 50 12.0

clc00597 55 0.0

Clkf06808 61 0.0 I. Campos et al. 17 SI

Cpr50Cbe02095 53 17.0

Cpr51Ae03998 56 1.8

Cpr62Bcf01099 65 0.0

Cpr64Ade02505 43 0.0

CSN3f02855 52 9.6

CSN7e02176 65 12.3 cv-cf04940 50 4.0 cv-cf07633 69 13.0

CycAc05304 50 2.0

Cyp4c3c06288 34 5.9 cypee03803 54 3.7 dc00148 93 4.3

Dcr-2f06544 50 0.0 deflc06100 52 9.6 dele02039 80 1.3

Dip-Cf00706 49 12.2

DNApol-α50f02992 114 14.9

DopEcRc02142 52 1.9 dreame00821 189 26.5 dsc01777 36 0.0 dsff00109 50 18.0 dyn-p25c02174 53 11.3 e02022 55 1.8

Eaat2e03003 22 9.1

Eaf6d06605 37 0.0

Ect4e03540 136 28.7

Ect4e03740 49 4.1 18 SI I. Campos et al.

Edg84Ae02715 48 12.5

eglEP938 55 1.8

eIF-1Ac04533 46 0.0

eIF2B-gammac01931 280 26.1

elkf00820 64 0.0

Elongin-Ce01107 51 2.0

empe04154 68 13.2

Enoe01615 58 3.4

Enof07543 51 11.8

EP1244EP1244 109 0.0

EP2404 32 6.3

EP2515 40 0.0

EP2520 59 0.0

EP3542 57 1.8

EP732 57 0.0

EP937 58 6.9

EP995EP995 63 0.0

Eps-15EP2513 19 5.3

escld01514 140 27.9

esnf00447 61 19.7

Ets21Cf03639 58 0.0

f04861 68 0.0

frede02229 60 10.0

garzEP2028 70 7.1

Gas41f05565 111 14.4

gishe01759 36 5.6

gktc03958 50 0.0 I. Campos et al. 19 SI

glof02674 221 59.7

Glu-RIBf01757 49 4.1 gogof05264 34 0.0

Gr28bc01884 92 6.5

Grip75f05483 43 0.0 grpe00087 171 43.9

Gs1lf02438 162 35.2

GstD3d06796 62 17.7

GstD9f06984 44 9.1

GstE3e00878 167 18.6

Gα49Bf04219 47 21.3

Gγ30Ae00834 108 25.0

Handf03901 0 ** hayf00028 55 0.0

Hel25Ee02545 48 22.9 herf03287 60 21.7

HerpEP2450 63 4.8

Heyf06656 34 0.0

HGTXd00083 0 *

Hnf4e02246 74 6.8

HopEP418 41 0.0

Hrb27Cf04375 0 ** htld07110 49 0.0

HtrA2f03785 58 17.2 ined07155 108 4.6 ire-1f02170 0 * itpEP2287 66 13.6 20 SI I. Campos et al.

jpd04563 69 5.8

k05816EP695 60 0.0

KaiRIAe01443 68 20.6

Khce02141 67 3.0

kkve03205 63 20.6

Klcc02312 167 18.0

knkf01902 37 13.5

krimpf06583 110 28.2

krzc01503 49 14.3

kstd11183 362 49.7

l(2)34FaEP2418 126 10.3

l(2)k07433e00176 56 3.6

leaEP2582 0 ***

lige04268 112 20.5

Liprin-αEP2141 or homerEP2141 63 0.0

lmgd09381 92 19.6

lolaEP2537 54 0.0

loqsf00791 51 2.0

Lrr47e00177 52 7.7

masc01899 40 5.0

matse03077 62 14.5

Mdhd11068 98 6.1

MED15f04180 58 8.6

MED20f00955 32 6.3

Mef2EP2002a 38 0.0

MESR3EP2221 57 5.3

Met75Caf00316 44 2.3 I. Campos et al. 21 SI

miltd03910 83 16.9 mip40f00474 50 0.0

Mkk4e01485 25 4.0 mole02670 60 18.3 morgueEP1184 91 17.6 mRpL1f05962 45 0.0 mRpL24f06692 48 16.7 mRpL43f05643 80 10.0 mRpL51c04791 66 3.0 mRpL9c00642 95 5.3 mRpS21e03199 57 3.5 mRpS28e02239 50 6.0 mRpS28f03356 52 0.0 mRpS33f01766 103 20.4

Mst36Fbf04849 116 24.1

MTA1-liked09140 39 5.1

NaCP60EEP348 or RpL41EP348 71 5.6 nAcRα-34Ef00872 30 16.7

Nckx30Ce00401 37 10.8

Nep4c02841 41 2.4 netf04249 53 1.9

Neu3c01955 229 48.5

NLazf03602 110 29.1 nocturnind05983 50 12.0 noif05442 48 8.3 notd06314 62 25.8

Notumd00939 47 14.9 22 SI I. Campos et al.

n-sybd02894 45 0.0

nxf2f05960 38 5.3

Nxt1f04855 57 10.5

olf413f02553 82 22.0

Optixc01718 63 0.0

Or35af02057 56 3.6

orf06278 40 0.0

Osi6f07607 0 **

otkEP2017 105 16.2

Pcaff02830 45 4.4

Pcaff05456 58 0.0

Pcf01890 311 54.7

PDCD-5c04145 52 15.4

Pde11e03811 59 13.6

Pde1cc04487 40 0.0

pdm3f00828 30 16.7

pgant4f02186 63 12.7

Picote02367 58 8.6

pief05500 74 4.1

Pkc98Ef06221 81 23.5

Plapd09025 56 3.6

plef01945 0 **

Pp2A-29BEP2332 52 9.6

prominin-likef03422 37 2.7

Prosα6EP2318 40 0.0

Prp18f05974 74 0.0

Ptp61Fc05292 56 1.8 I. Campos et al. 23 SI

Ptpmegf01047 or methl10f01047 47 0.0 pugc03481 44 4.5 qkr54Be02070 121 9.9

Rab1e01287 62 11.3

Rab39GG01805 62 0.0

Rab6EP2397 38 0.0

Rad1f02242 107 26.2

RanGapEP1173 53 11.3

Rapgap1e04634 47 4.3

Rca1e02614 42 9.5

Rdlf02994 93 25.8

Repf01510 63 0.0 reptf01801 42 0.0 rho-5c01455 66 0.0

RhoGAP100Fc01128 61 0.0

RhoGEF2e03784 83 14.5

RluA-2EP2102 58 12.1

Roc2EP2487 46 4.3

Rpb11c01686 47 17.0

Rpn11f00386 67 26.9

RpS15e01611 60 15.0

Rpt1EP2153 or CG17985EP2153 47 6.4

Rrp1c00695 43 4.7

SAKc06612 45 15.6

Samuele02949 59 20.3

Sbc02563 0 *

Scrf05078 52 9.6 24 SI I. Campos et al.

SdhBc00364 37 5.4

sds22e00975 62 21.0

sec10f03085 77 1.3

sec31c02461 51 11.8

sensf06181 80 22.5

Ser12f03416 212 43.9

serpe02821 63 12.7

Shalf00495 61 9.8

sidef00677 130 59.2

SMC2f06842 46 8.7

sobd06074 49 14.3

sod09734 84 6.0

spictEP2202 36 0.0

spiEP2378 60 0.0

Spn1f02145 113 23.0

Spn5c01214 108 22.2

spn-Ec00786 73 27.4

Src64Bc04709 65 3.1

Ssb-c31ae02272 63 4.8

staic01639 62 21.0

Stame00677 183 43.2

Strn-Mlckc02860 105 22.9

su(Hw)e04061 0 *

Sur-8e02803 81 12.3

Sure00744 69 14.5

Surf4d04274 64 1.6

synje02597 51 11.8 I. Campos et al. 25 SI

Syx5EP2313 51 5.9

Taf6f06930 139 28.8

Tbpf00190 54 0.0

T-cp1c03987 0 *

TepIIId03976 73 1.4 texd08549 101 25.7

TFAMc01716 32 3.1

TfIIA-Ld08487 70 1.4

TfIIEalphae01382 59 1.7

Tkf06233 or Ect3f06233 121 28.9

TkrEP1162 40 0.0

Top3αEP2272 47 6.4

Toporsf05115 36 27.8

TotCf01700 228 21.5

Traf1EP578 138 0.0 trxe00972 55 9.1

Tsc1f01910 58 6.9 tupd03613 0 **

Ubxd00281 66 13.6

UGPd07256 96 11.5

UGPf03515 56 0.0

Ugt58Fac05973 64 1.6

Ugt86Die00862 58 12.1

UK114e03986 45 6.7 unc-104d11204 51 3.9 undEP424 46 4.3

Urof04888 68 1.5 26 SI I. Campos et al.

ushf06555 0 **

Vangf04290 47 17.0

vgf02736 0 **

Vha16EP2372 54 11.1

Vha68-2EP2364 63 3.2

wlsc06300 52 19.2

Wwoxf04545 82 14.6

yellow-e3e01012 112 25.9

εCOPEP2027 59 22.0

ζCOPe00020 0 **

655 insertional mutant lines obtained from Bloomington Stock Centre and re- balanced with CyO-CTG or TM3-TTG, depending on insertion site, were screened for wound healing phenotypes as described in Material and Methods. Number of embryos analysed (n) and percentage of opened wounds approximately 16 hours post wounding (% holes) are depicted. *- lines impossible to balance with CyO-CTG or TM3-TTG, depending on insertion site; **- lines impossible to wound or score due to strong morphological defects; ***- contaminated stocks